1 U.S. Horticultural Research Laboratory Ft. Pierce, Florida Breeding Citrus for HLB Resistance Kim Bowman Greg McCollum Randy Niedz Robert Shatters Ed Stover Ute Albrecht Lesley Benyon Randy Driggers Mizuri Hert Ric Stange YongPing Duan David Hall Richard Lee William Belknap
2 ARS Citrus Improvement The oldest citrus breeding program in the world W. T. Swingle H.J. Webber Sub-Tropical Laboratory Eustis, Fla. Swingle and Webber >75% of US citrus industry has rootstock and/or scion from USDA program. mainly rootstocks!
3 USDA Citrus Scion Releases Year Release Pedigree 1930 Minneola Orlando Duncan x Dancy 1959 Robinson Osceola Clementine x Orlando Lee 1963 Page Clementine x Minneola 1964 Nova Clementine x Orlando 1979 Sunburst Robinson x Osceola 1987 Fallglo (Clementine x Orlando) x Temple 1987 Flame Nucellar sport of Ruby Red 1987 Midsweet Nucellar sport of Homosassa 1989 Ambersweet (Clementine x Orlando) x midseason orange 2009 US Seedless Pineapple Irradiated Pineapple 2009 US Early Pride Irradiated Fallglo
4 Earlier USDA Released Rootstock Varieties Variety Year Industry % Carrizo/Troyer citrange Swingle citrumelo Sun Chu Sha mandarin 1988 < 1%
5 New Rootstocks from USDA US-852 released in 1999 US-812 released in 2001 US-802 released in 2007 US-897 released in 2007 US-942 for release in 2010
6 Focus on Developing HLB-and ACP Resistant Citrus HLB likely the single greatest threat to citrus No strong HLB resistance has been identified in cultivated Citrus scion varieties Transgenics appear to be the only medium term solution for strong HLB resistance Goal: add genes to reduce survival, growth, and/or virulence of causal pathogen, genes to deter psyllid vector, possibly suppress host disease response With little known about host /pathogen interaction, antimicrobial peptides have been a major focusmay also confer resistance to canker and CVC Other types of genes also being explored..
7 Harnessing nature s genetic engineer: Agrobacterium tumefaciens A. tumefaciens causes crown gall disease in many plant species By removing At genes for growth regulators and replacing with: 1) promoter 2) gene of interest etc. 3) gene for selectable marker (antibiotic) can express genes when and where you want, without gall formation. Image:
8 Citrus Transformation: Major Focus for HLB and Canker Resistance Transformation Selection Regeneration Evaluation Grow out Micro-grafting
9 Transgenic Project: Parallel Tracks 1 1a 2 3 2a 3a mm 4a Shoot-tip filter paper platform liquid medium Fastest track- possible home run using best available technology on rootstocks, sweet orange and grapefruit- high throughput. Goal is earliest possible resistant variety in field Emphasizing components which are deregulated in crop plants Experiments to overcome transformation bottlenecks Identifying new targets for transgenes Exploring other promoters, constructs, etc. first with easily transformed rootstock types
10 Antimicrobial Peptides Broadly active against groups of micro-organisms First line of active defense to combat infection in multicellular organisms Most are very small molecules, MAY move systemically Results in microbial death or prevents growth
11 In Vitro AMP Screening Agrobacterium and Sinorhizobium are related to Liberibacter Also using Xanthomonas citri Best AMPs, including D4E1 are effective in 1 µm range
12 Other Transgenes for HLB Resistance Transmembrane protein from Liberibacter is a target identified from the Liberibacter asiaticus genome (working with Duan group at USHRL) Phloem-specific protein induced during HLB infection (Bowman data USHRL) Working with ARS scientists in California to develop constructs so that ALL inserted genes are from Citrus!
13 300 independent scion (Hamlin, Midsweet, Valencia, and Ray Ruby) and >3500 independent rootstock regenerants established as micrografts
14 Transgenic Project: Challenging with HLB etc. 1 1a 2 3 2a 3a mm 4a Shoot-tip filter paper platform liquid medium Lots of plants of trifoliate hybrids transformed with D35S/D4E1 have been challenged No major differences, so far, in HLB symptoms or Liberibacter levels. D4E1-GE plants have better growth. Now using more active promoters and phloem specific promoters as well as different transgenes d35s:gus Activity in most tissue SS:GUS activity limited to phloem!
15 D4E1Hamlin challenged with CBC Control D4E1 Transformed Plant # 88 D4E1 Transformed Plant # 161
16 HLB-resistance from distant citrus and relatives reports of HLB resistance in different species related to citrus broad search may yield greater resistance Experiments include more than 50 genotypescollaboration Riverside Repository May identify genes which can then be used to transform commercial citrus Citrus gene that should permit flowering within year of seed germination, permitting rapid use of genes from distant relatives
17 Carrizo transformed with D35S:: Citrus FT Gloria Moore- UF Horticulture
18 Evaluating varieties for HLB-tolerance Appears that citrus varieties vary widely in their rate of developing HLB Identified 8 groves in the Indian River area reporting presence of HLB and multiple specialty cultivars Avoided reported edge effects and bias, randomly sampled 20 trees /cultivar /grove Ran q-pcr using 16S CLas rdna Wenbin primers (APHIS standard); 760 trees were sampled
19 Random sampling of interior trees in commercial groves with multiple specialty cultivars: % of 20 trees with Ct <36 from q-pcr of CLas rdna (April 2010) Cultivar Indian River Martin 1 Martin 2 St. Lucie 1 St. Lucie 2 St. Lucie 3 St. Lucie 4 Sweet orange Grapefruit Sunburst Palm Beach Temple Fallglo Minneola Murcott Ortanique 30
20 ANOVA on qpcr Ct data for CLas 16S rdna for citrus cultivars in 6 Indian River area groves: April The lower the Ct value, the higher the CLas titer. (Data excluded for the 2 groves with no CLasdetectedin sweet orange) Mean Ct grove Mean # CLas % trees N Ct normalized genome/sample* Ct<36 Minneola d -2.4 c % Murcott cd -1.3 bc % Sweet orange bc -0.4 b % Grapefruit ab 1.1 a 40 20% Temple a 1.3 a 9 15% Fallglo a 1.5 a 13 18% Sunburst a 1.6 a %
21 Issues regarding HLB/ACP-tolerance If resistance or tolerance is confirmed, how may this benefit citrus industries? How much of a delay in symptom development / compromise of cropping is needed to be useful? Could resistant/tolerant types be used to protect more susceptible cultivars? NOT suggesting we all grow Temple! If significant U1 resistance/tolerance is found in conventional cultivars, may find in related hybrids that are very sweet-orange or grapefruit like As well as possibly identifying genes for intragenic use
22 Diapositiva 21 U1 REd text: Confusing USHRL, 02/06/2010
24 Thanks! Florida Citrus Production Research Advisory Council New Varieties Development and Management Corporation Florida Citrus Research Foundation (Whitmore) Jodi Avila Scott Ciliento Daniel Davis Emily Domagtoy Tim Lind Mike Rutherford Lindsay Turnbull Eldridge Wynn Wayne Brown Regina Conley Jacqueline Depaz Lorri Hutchinson Jerry Mozuruk James Salvatore Kerry Worton